Light Sheet Fluorescence Microscopy Research Articles

Mantis: high-throughput 4D imaging and analysis of the molecular and physical architecture of cells.

High-throughput dynamic imaging of cells and organelles is essential for understanding complex cellular responses. We report Mantis, a high-throughput 4D microscope that integrates two complementary, gentle, live-cell imaging technologies: remote-refocus label-free microscopy and oblique light-sheet fluorescence microscopy. Additionally, we report shrimPy, an open-source software for high-throughput imaging, deconvolution, and single-cell phenotyping of 4D data. Using Mantis and shrimPy, we achieved high-content correlative imaging of molecular dynamics and the physical architecture of 20 cell lines every 15 minutes over 7.5 hours. This platform also facilitated detailed measurements of the impacts of viral infection on the architecture of host cells and host proteins. The Mantis platform can enable high-throughput profiling of intracellular dynamics, long-term imaging and analysis of cellular responses to perturbations, and live-cell optical screens to dissect gene regulatory networks.

Improved image contrast in nonlinear light-sheet fluorescence microscopy using i2\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$^2$$\\end{document}PIE Pulse compression

Nonlinear microscopy has become an invaluable tool for biological imaging, offering high-resolution visualization of biological specimens. In this manuscript, we present the application of a spectral phase measurement technique, i2\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$^2$$\\end{document}PIE, to compress broad-bandwidth supercontinuum pulses for two-photon excitation fluorescence light-sheet fluorescence microscopy. The results demonstrated a significant improvement in the two-photon excitation response achieved. We also showed that the implementation of i2\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$^2$$\\end{document}PIE allowed for enhanced image contrasts when compared to conventional compression techniques, with i2\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$^2$$\\end{document}PIE producing an image contrast improvement over conventional methods by over 50%.

Intelligent Beam Optimization for Light-Sheet Fluorescence Microscopy through Deep Learning

Intelligent Beam Optimization for Light-Sheet Fluorescence Microscopy through Deep Learning

Multispectral imaging for characterizing autofluorescent tissues

Selective Plane Illumination Microscopy (SPIM) has become an emerging technology since its first application for 3D in-vivo imaging of the development of a living organism. An extensive number of works have been published, improving both the speed of acquisition and the resolution of the systems. Furthermore, multispectral imaging allows the effective separation of overlapping signals associated with different fluorophores from the spectrum over the whole field-of-view of the analyzed sample. To eliminate the need of using fluorescent dyes, this technique can also be applied to autofluorescence imaging. However, the effective separation of the overlapped spectra in autofluorescence imaging necessitates the use of mathematical tools. In this work, we explore the application of a method based on Principal Component Analysis (PCA) that enables tissue characterization upon spectral autofluorescence data without the use of fluorophores. Thus, enabling the separation of different tissue types in fixed and living samples with no need of staining techniques. Two procedures are described for acquiring spectral data, including a single excitation based method and a multi-excitation scanning approach. In both cases, we demonstrate the effective separation of various tissue types based on their unique autofluorescence spectra.

Beam shaping in light-sheet microscopy: an experimental analysis

Abstract Thanks to its unique optical sectioning capability, light-sheet fluorescence microscopy has proven to be a powerful technique for volumetric imaging of entire model organisms with high spatial and temporal resolution. For light sheet generation with scanned laser beams, holographic beam shaping offers precise control over the optical fields exciting the fluorescence. Various illumination schemes have been proposed, aiming for best image quality with regard to axial resolution, optical sectioning, illumination homogeneity and photobleaching while at the same time retaining a large field of view. Here, we have engineered and characterized a variety of beams and analyzed their imaging performance by using phantom samples and zebrafish embryos. These data may assist researchers to select the light sheet best suited to the imaging application at hand.

High-speed light-sheet fluorescent microscopy with an adaptive three-dimensional region-of-signal identification method

Light-sheet fluorescent microscopy (LSFM) is a crucial 3D imaging technique for tissues with advantages in high 3D spatial resolution, effective optical sectioning, and quick imaging. Prior to imaging, the sample's imaging depth, imaging region, and region of interest (ROI) division should all be established in accordance with the sample's outermost dimensions. However, because biological samples are inherently irregular a traditional imaging flow that images ROI by ROI, slice by slice, would waste a great deal of time and data storage space by additionally recording non-signal regions. In order to solve this problem, we create a high-speed LSFM with an adaptive three-dimensional region-of-signal identification method (RSI). We immediately perform crude imaging for each ROI before the software analyzes the longitudinal distribution and, ultimately, uses dimensionality reduction, binarization, filtering, and redundancy expansion to acquire the signal region result. By imaging two mouse spinal cord segments with a total of 170 ROIs and 180 ROIs on two channels, respectively, the high-speed imaging capability is validated. Our method reduced the overall imaging time and raw data size by more than treble and 14 times, respectively, without losing signal. With a programmable imaging range based on the real signal region, this method offers an efficient tool for flexible light-sheet microscopy. It can also be applied to other microscopy techniques to speed up imaging.

A New Cell Type in Pulmonary Veins

Pulmonary veins (PVs) carry oxygenated blood from lungs back to the heart and may play an important role in preventing blood backflow from the left atrium. Previous studies show that large PVs exhibit spontaneously pulsatile contractions in cardiomyocyte-like manner. However, the contractions of small PVs have not been investigated. We hypothesized that Ca2+ signaling mechanisms in the myocytes mediate small PV contractions. Small, 4th-order PVs from C57BL6 mice were obtained and imaged. Pressurized PVs showed ryanodine receptor-dependent Ca2+ signals and spontaneous contractions. Light sheet fluorescence microscopy and immunostaining showed the expression of troponin C, a cardiomyocyte marker, in the myocyte layer of small PVs, which was not observed in the pulmonary arteries, vena cava, or mesenteric veins. Based on these results, we termed these cells cardiomyocyte-like (CML) cells. In the study of mice with Myh11 Cre-driven tdTomato expression, Myh11 expression was observed in CML cells in small PVs, which was not seen in the smooth muscle cells (SMC) of small PAs. Endothelial protrusions crossing the connective tissue layer into the CML layer were also observed in PVs. Previous studies showed PV remodeling in pulmonary hypertension (PH), and impaired Ca2+ signaling in PAs contribute to higher pulmonary arterial pressure in PH. We further hypothesized that ryanodine receptor (RyR) Ca2+ signals in CML cells are impaired in PH. Results show that RyR Ca2+ signaling was reduced in CML cells of PH mice (injected with Su5416, subjected to chronic hypoxic conditions). Overall, our findings identify a new cell type in small PVs. Moreover, our data show that RyR Ca2+ signals in CML cells underlie spontaneous contractions in small PV, and are impaired in PH. We propose that RyR signaling mechanisms in CML cells may be possible targets for therapy in pulmonary vascular disorders. This work was supported by funding from the National Institute of Health to Swapnil Sonkusare (R01:HL146914, R01:HL157407). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Unraveling the Role of Enkephalin Signaling in Respiratory Control

Opioid-induced respiratory depression (OIRD) is the primary cause of death during opioid overdose characterized by a dramatic decrease in respiratory frequency and regularity. Opioid drugs cause neural depression in the ventral respiratory column (VRC) of the medulla via activation of mu-opioid receptors (MORs) that partially contribute to breathing cessation. Nonetheless, little is known about the role of endogenous opioid signaling in respiratory modulation. This study investigates the role of endogenous MORs ligands, enkephalins encoded by the Penk gene, in breathing control. To test whether enkephalin-expressing neurons of the preBӧtzinger Complex (preBӧtC) — the inspiratory rhythm-generating region of the VRC — are necessary for breathing rhythmogenesis, we permanently silenced the region’s Penk+ population via AAV1 injections encoding tetanus toxin light chain. Using whole-body plethysmography, we recorded the animals' respiratory performance within 14 days post-injection. During the second week of testing, we observed unexpected mortality in some experimental but not control animals; post-hoc analysis of breathing traces revealed the development of severe tachypnea in succumbed mice. These data suggest that Penk+ preBӧtC cells are integral for normal respiration. Still, these observations did not assess the specific contribution of enkephalin, as opposed to other types of signaling, to breathing stability. To directly dissect the effects of enkephalin on respiratory rhythm, we utilized optogenetics and pharmacological approaches in brainstem slice preparation from neonatal mice. Brief optogenetic activation of Penk+ neurons in the preBӧtC evoked population bursts while prolonged stimulation increased the rhythm frequency. However, the application of MOR antagonist Naloxone did not affect the network’s responses to light stimulations indicating the lack of MOR/enkephalin involvement in mediating these excitatory effects. High fidelity and temporal coordination of evoked bursts implicated glutamatergic transmission as the primary mechanism of excitation. Likewise, we were able to optogenetically drive respiratory rhythm in urethane-anesthetized spontaneously breathing adult mice by stimulating the preBӧtC. Further, there was a small but significant difference in breathing responses in the presence of Naloxone compared to control conditions. We hypothesized that longitudinal projections to the VRC that might be disrupted during slice preparation contributed enkephalin to the respiratory circuits in the whole-body preparation. We utilized recombinase-dependent reporter mice crosses and retrograde AAV tracers to identify brain-wide Penk inputs to the preBӧtC. Using conventional and light sheet fluorescence microscopy, we located regions in the pons, midbrain, and forebrain that send direct enkephalinergic projections to the VRC. Functional characterizations of these projections will potentially uncover state-dependent mechanisms of respiratory modulation via endogenous opioids. Untimely, this study will help elucidate the role of enkephalin signaling in breathing control and may establish therapeutic targets for preventing OIRD. R01HL166317, R00HL145004. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A vascularized breast cancer spheroid platform for the ranked evaluation of tumor microenvironment-targeted drugs by light sheet fluorescence microscopy

Targeting the supportive tumor microenvironment (TME) is an approach of high interest in cancer drug development. However, assessing TME-targeted drug candidates presents a unique set of challenges. We develop a comprehensive screening platform that allows monitoring, quantifying, and ranking drug-induced effects in self-organizing, vascularized tumor spheroids (VTSs). The confrontation of four human-derived cell populations makes it possible to recreate and study complex changes in TME composition and cell-cell interaction. The platform is modular and adaptable for tumor entity or genetic manipulation. Treatment effects are recorded by light sheet fluorescence microscopy and translated by an advanced image analysis routine in processable multi-parametric datasets. The system proved to be robust, with strong interassay reliability. We demonstrate the platform’s utility for evaluating TME-targeted antifibrotic and antiangiogenic drugs side-by-side. The platform’s output enabled the differential evaluation of even closely related drug candidates according to projected therapeutic needs.

A vascularized in vivo melanoma model suitable for metastasis research of different tumor stages using fundamentally different bioinks

Although 2D cancer models have been the standard for drug development, they don't resemble in vivo properties adequately. 3D models can potentially overcome this. Bioprinting is a promising technique for more refined models to investigate central processes in tumor development such as proliferation, dormancy or metastasis.We aimed to analyze bioinks, which could mimic these different tumor stages in a cast vascularized arteriovenous loop melanoma model in vivo. It has the advantage to be a closed system with a defined microenvironment, supplied only with one vessel—ideal for metastasis research.Tested bioinks showed significant differences in composition, printability, stiffness and microscopic pore structure, which led to different tumor stages (Matrigel and Alg/HA/Gel for progression, Cellink Bioink for dormancy) and resulted in different primary tumor growth (Matrigel significantly higher than Cellink Bioink). Light-sheet fluorescence microscopy revealed differences in vascularization and hemorrhages with no additional vessels found in Cellink Bioink. Histologically, typical human melanoma with different stages was demonstrated. HMB-45-positive tumors in progression inks were infiltrated by macrophages (CD163), highly proliferative (Ki67) and metastatic (MITF/BRN2, ATX, MMP3). Stainings of lymph nodes revealed metastases even without significant primary tumor growth in Cellink Bioink.This model can be used to study tumor pathology and metastasis of different tumor stages and therapies.

Interferometric modulation for generating extended light sheet: improving field of view.

Light-sheet fluorescence microscopy (LSFM) has emerged as a powerful and versatile imaging technique renowned for its remarkable features, including high-speed 3D tomography, minimal photobleaching, and low phototoxicity. The interference light-sheet fluorescence microscope, with its larger field of view (FOV) and more uniform axial resolution, possesses significant potential for a wide range of applications in biology and medicine. The aim of this study is to investigate the interference behavior among multiple light sheets (LSs) in LSFM and optimize the FOV and resolution of the light-sheet fluorescence microscope. We conducted a detailed investigation of the interference effects among LSs through theoretical derivation and numerical simulations, aiming to find optimal parameters. Subsequently, we constructed a customized system of multi-LSFM that incorporates both interference light sheets (ILS) and noninterference light-sheet configurations. We performed beam imaging and microsphere imaging tests to evaluate the FOV and axial resolution of these systems. Using our custom-designed light-sheet fluorescence microscope, we captured the intensity distribution profiles of both interference and noninterference light sheets (NILS). Additionally, we conducted imaging tests on microspheres to assess their imaging outcomes. The ILS not only exhibits a larger FOV compared to the NILS but also demonstrates a more uniform axial resolution. By effectively modulating the interference among multiple LSs, it is possible to optimize the intensity distribution of the LSs, expand the FOV, and achieve a more uniform axial resolution.

Visualisation of gene expression within the context of tissues using an X-ray computed tomography-based multimodal approach

The development of an organism is orchestrated by the spatial and temporal expression of genes. Accurate visualisation of gene expression patterns in the context of the surrounding tissues offers a glimpse into the mechanisms that drive morphogenesis. We developed correlative light-sheet fluorescence microscopy and X-ray computed tomography approach to map gene expression patterns to the whole organism`s 3D anatomy. We show that this multimodal approach is applicable to gene expression visualized by protein-specific antibodies and fluorescence RNA in situ hybridisation offering a detailed understanding of individual phenotypic variations in model organisms. Furthermore, the approach offers a unique possibility to identify tissues together with their 3D cellular and molecular composition in anatomically less-defined in vitro models, such as organoids. We anticipate that the visual and quantitative insights into the 3D distribution of gene expression within tissue architecture, by multimodal approach developed here, will be equally valuable for reference atlases of model organisms development, as well as for comprehensive screens, and morphogenesis studies of in vitro models.

An MR-based brain template and atlas for optical projection tomography and light sheet fluorescence microscopy in neuroscience.

Optical Projection Tomography (OPT) and light sheet fluorescence microscopy (LSFM) are high resolution optical imaging techniques, ideally suited for ex vivo 3D whole mouse brain imaging. Although they exhibit high specificity for their targets, the anatomical detail provided by tissue autofluorescence remains limited. T1-weighted images were acquired from 19 BABB or DBE cleared brains to create an MR template using serial longitudinal registration. Afterwards, fluorescent OPT and LSFM images were coregistered/normalized to the MR template to create fusion images. Volumetric calculations revealed a significant difference between BABB and DBE cleared brains, leading to develop two optimized templates, with associated tissue priors and brain atlas, for BABB (OCUM) and DBE (iOCUM). By creating fusion images, we identified virus infected brain regions, mapped dopamine transporter and translocator protein expression, and traced innervation from the eye along the optic tract to the thalamus and superior colliculus using cholera toxin B. Fusion images allowed for precise anatomical identification of fluorescent signal in the detailed anatomical context provided by MR. The possibility to anatomically map fluorescent signals on magnetic resonance (MR) images, widely used in clinical and preclinical neuroscience, would greatly benefit applications of optical imaging of mouse brain. These specific MR templates for cleared brains enable a broad range of neuroscientific applications integrating 3D optical brain imaging.

Distortion Correction and Denoising of Light Sheet Fluorescence Images.

Light Sheet Fluorescence Microscopy (LSFM) has emerged as a valuable tool for neurobiologists, enabling the rapid and high-quality volumetric imaging of mice brains. However, inherent artifacts and distortions introduced during the imaging process necessitate careful enhancement of LSFM images for optimal 3D reconstructions. This work aims to correct images slice by slice before reconstructing 3D volumes. Our approach involves a three-step process: firstly, the implementation of a deblurring algorithm using the work of K. Becker; secondly, an automatic contrast enhancement; and thirdly, the development of a convolutional denoising auto-encoder featuring skip connections to effectively address noise introduced by contrast enhancement, particularly excelling in handling mixed Poisson-Gaussian noise. Additionally, we tackle the challenge of axial distortion in LSFM by introducing an approach based on an auto-encoder trained on bead calibration images. The proposed pipeline demonstrates a complete solution, presenting promising results that surpass existing methods in denoising LSFM images. These advancements hold potential to significantly improve the interpretation of biological data.

Abstract 4220: Combined light sheet and multispectral imaging facilitates the detection of tumor-associated tertiary lymphoid structures in the KP NSCLC model

Abstract Tertiary lymphoid structures (TLS) have recently emerged as a predictive biomarker in cancer immunotherapy. Their consideration in preclinical drug development is impeded by rare and transient appearance and random distribution, making them difficult to analyze with conventional sampling methods. We applied whole-organ tissue clearing and 3D staining of B and T cell clusters to comprehensively identify TLS in a KP GEMM of NSCLC using 3D Light Sheet Fluorescence Microscopy (3D LSFM). To further investigate cellular composition of TLS we performed 3D LSFM-educated sectioning of relevant ROIs and cyclic immunofluorescence of the same samples. 3D LSFM facilitated correlation of tumor features and location with T cell infiltration as well as the analysis of number, size and location of TLS. The majority of TLS were located in proximity to bronchi instead of intratumorally. LSFM-guided multiplex immunofluorescence revealed further details on cellular composition and maturation status of TLS. This combination of 3D and 2D imaging modalities offers a holistic approach for the identification of TLS in whole murine tumors or organs. The technology could help studying effects of immunotherapy on de novo formation or maturation of TLS. Citation Format: Nils O'Brien, Birte Appelt, Marie-Lena Moerbitz, Joerg P. Mueller, Frank Herting, Pablo Umaña, Claudio Murgia, Sara Colombetti, Thomas Pöschinger. Combined light sheet and multispectral imaging facilitates the detection of tumor-associated tertiary lymphoid structures in the KP NSCLC model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4220.

Oxidation–reduction imaging of myoglobin reveals two-phase oxidation in the reperfused myocardium

Myocardial infarction (MI) is a serious acute cardiovascular syndrome that causes myocardial injury due to blood flow obstruction to a specific myocardial area. Under ischemic–reperfusion settings, a burst of reactive oxygen species is generated, leading to redox imbalance that could be attributed to several molecules, including myoglobin. Myoglobin is dynamic and exhibits various oxidation–reduction states that have been an early subject of attention in the food industry, specifically for meat consumers. However, rarely if ever have the myoglobin optical properties been used to measure the severity of MI. In the current study, we develop a novel imaging pipeline that integrates tissue clearing, confocal and light sheet fluorescence microscopy, combined with imaging analysis, and processing tools to investigate and characterize the oxidation–reduction states of myoglobin in the ischemic area of the cleared myocardium post-MI. Using spectral imaging, we have characterized the endogenous fluorescence of the myocardium and demonstrated that it is partly composed by fluorescence of myoglobin. Under ischemia–reperfusion experimental settings, we report that the infarcted myocardium spectral signature is similar to that of oxidized myoglobin signal that peaks 3 h post-reperfusion and decreases with cardioprotection. The infarct size assessed by oxidation–reduction imaging at 3 h post-reperfusion was correlated to the one estimated with late gadolinium enhancement MRI at 24 h post-reperfusion. In conclusion, this original work suggests that the redox state of myoglobin can be used as a promising imaging biomarker for characterizing and estimating the size of the MI during early phases of reperfusion.

Expansion and Light-Sheet Microscopy for Nanoscale 3D Imaging.

Expansion Microscopy (ExM) and Light-Sheet Fluorescence Microscopy (LSFM) are forefront imaging techniques that enable high-resolution visualization of biological specimens. ExM enhances nanoscale investigation using conventional fluorescence microscopes, while LSFM offers rapid, minimally invasive imaging over large volumes. This review explores the joint advancements of ExM and LSFM, focusing on the excellent performance of the integrated modality obtained from the combination of the two, which is refer to as ExLSFM. In doing so, the chemical processes required for ExM, the tailored optical setup of LSFM for examining expanded samples, and the adjustments in sample preparation for accurate data collection are emphasized. It is delve into various specimen types studied using this integrated method and assess its potential for future applications. The goal of this literature review is to enrich the comprehension of ExM and LSFM, encouraging their wider use and ongoing development, looking forward to the upcoming challenges, and anticipating innovations in these imaging techniques.

Recent developments in biomedical applications of metasurface optics

Metasurfaces have attracted considerable attention because of their unique optical capabilities to control the fundamental properties of light, such as amplitude, phase, and polarization. The flat nature of metasurfaces can help reduce the complexities and bulk of conventional optical systems. After a decade of rapid progress, metasurfaces are close to maturity and have found their role in various optical applications. This review emphasizes the significant advancements and emerging applications of metasurfaces in biomedical optics, particularly focusing on beam shaping for laser treatments, light-sheet fluorescence microscopy, HiLo microscopy, and optical trapping. Looking forward, we discuss foreseeable challenges for integrating metasurfaces into biomedical, preclinical, and clinical systems.

From breast cancer cell homing to the onset of early bone metastasis: The role of bone (re)modeling in early lesion formation.

Breast cancer often metastasizes to bone, causing osteolytic lesions. Structural and biophysical changes are rarely studied yet are hypothesized to influence metastasis. We developed a mouse model of early bone metastasis and multimodal imaging to quantify cancer cell homing, bone (re)modeling, and onset of metastasis. Using tissue clearing and three-dimensional (3D) light sheet fluorescence microscopy, we located enhanced green fluorescent protein-positive cancer cells and small clusters in intact bones and quantified their size and spatial distribution. We detected early bone lesions using in vivo microcomputed tomography (microCT)-based time-lapse morphometry and revealed altered bone (re)modeling in the absence of detectable lesions. With a new microCT image analysis tool, we tracked the growth of early lesions over time. We showed that cancer cells home in all bone compartments, while osteolytic lesions are only detected in the metaphysis, a region of high (re)modeling. Our study suggests that higher rates of (re)modeling act as a driver of lesion formation during early metastasis.

Super-sectioning with multi-sheet reversible saturable optical fluorescence transitions (RESOLFT) microscopy

Light-sheet fluorescence microscopy is an invaluable tool for four-dimensional biological imaging of multicellular systems due to the rapid volumetric imaging and minimal illumination dosage. However, it is challenging to retrieve fine subcellular information, especially in living cells, due to the width of the sheet of light (>1 μm). Here, using reversibly switchable fluorescent proteins (RSFPs) and a periodic light pattern for photoswitching, we demonstrate a super-resolution imaging method for rapid volumetric imaging of subcellular structures called multi-sheet RESOLFT. Multiple emission-sheets with a width that is far below the diffraction limit are created in parallel increasing recording speed (1–2 Hz) to provide super-sectioning ability (<100 nm). Our technology is compatible with various RSFPs due to its minimal requirement in the number of switching cycles and can be used to study a plethora of cellular structures. We track cellular processes such as cell division, actin motion and the dynamics of virus-like particles in three dimensions.